Glioblastomas (GBMs) are one of the most difficult cancers to treat. Their success in defying therapy relies in large part on their dynamic cellular heterogeneity and on their ability to subvert their environment, in particular to create an immune suppressive milieu. This project will address the role of the newly discovered communication vehicles in cancer, extracellular vesicles (EVs) which can carry RNA and protein as informational molecules. Our goal is to evaluate to what extent EVs produced by these tumors contribute to an immune suppressive environment through their interaction with tumor-associated macrophages and microglia (TAMs), and to what extent EVs produced by different genetic subtypes of gliomas, and the stem-like cells within these tumors, underlie to their ability to re-define themselves and escape therapy. In Aim 1 we will assess EV-mediated information transfer between glioma tumors and microglia/macrophages in the tumor microenvironment and its effect on immune suppression. In Aim 2 we will evaluate the role of EVs in modulating GBM stem-like cell (GSC) phenotypes and therapeutic resistance. Studies will use syngeneic mouse glioma models as well as human GBM stem-like neurosphere cultures and patient-derived xenograft in mouse models developed with Core C. We will generate an array of vectors to label cells with multiple luciferases and fluorescent proteins, as well as create capsules to contain tumor cells while allowing release of EVs and/or small molecules within the brain. FACS analysis and RNAseq will be used to define the transcriptome of different cell populations in the brain. Biofluids from mice, as well as from GBM patients (Core B), will be sent to Project 2 for protein analysis. Correlative analysis of mRNA/ protein contents and GBM genotypes will be assessed, as well as how EV contents reflect changes in GBM subtype and resistance to therapy. Our findings will be integrated with those of Project 3 in the context of preclinical evaluation of Gene- Mediated Cytotoxic Immunotherapy (GMIC) combined with immune checkpoint inhibition. Organizational oversight of rigor and reproducibility will be provided by Core A. Studies in this project will serve to inform the development of the projected clinical trial by elucidating how EVs influence and report on GBM status, and how they can be manipulated to improve this type of immunotherapy in the context of the GBM brain tumors.